Study On The Interaction Between Some Medicinal Molecules And Biomacromolecules

Protein anddeoxyribonucleic acid(DNA) are the basic composition of all organism, they play important roles in all kinds of life activities. DNA is the carrier of genetic materials which acts the main role to the biological evolution, and protein is the expresser of these genetic materials. So, there have been an interesting research field of life sciences, pharmcodynitics and chemistry of them. The investigation of interaction between small medicinal molecules with biomacromolecules such as bovine serum albumin(BSA), human serum albumin(HAS) or DNA is helpful for us to understand the mechanism of drugs, it is also helpful to us to exploit new drugs which would be more effective to diseases and less side-effect to health. In this dissertation, on the basis of the previous research, the fluorescence and UV-visible spectrophotometry were used to investigate several medicinal molecules interact with BSA, HSA and DNA. The thesis consists of six parts as follows:Chapter 1. The summarization of research on interaction of medicinal molecules and biomacromoleculesIn this section, the development of the research on the interaction between medicinal molecules and biomacromolecules have been expatiated in recent years. The structure and the function of BSA, HSA and DNA was introduced, and the methods of this research field were summarized especially the methods of fluorescence and UV-visible spectrophotometry, so the theory was gainable for the development of this thesis.Chapter 2. Studies on the interaction between icariin and human serum albumin by fluorescence spectrometryIcariin(ICA) is one of the main components of a traditional Chinese herbal medicine which named Epimedium. In this section, the interaction between icariin and human serum albumin in physiological condition Was studied by fluorescence and UV-visible spectrophotometry. It is obviously that the fluorescence of HSA could be quenched by ICA, so it is clearly that there is strong binding between icariin and HSA. The binding mechanism was proved to be the single static quenching process according to the deescalating binding constant along with the escalating temperatures (29℃and 42℃), the binding constant was measured to be 3.80×10⁴ L·mol^-1 at 29℃. The hydrophobic forces plays a major role in the reaction according to the thermodynamic parametersâ–³H,â–³G andâ–³S(â–³H=11.18 kJ·mol^-1,â–³G=-26.46 kJ·mol^-1＜0,â–³S=124.64 J·mol^-1·K^-1＞0). In addition, the binding distance between donor(HSA) and acceptor(ICA) was calculated to be 4.18nm which based on the theory of Forster spectroscopy energy transfer, the energy transfer efficiency (E=0.07898) and the overlap integral of the fluorescence emission spectrum of HSA and the absorption spectrum of ICA(J=1.78×10¹⁴cm³Â·L·mol^-1). The study of synchronous fluorescence spectra and the three-dimensional fluorescence spectra was used to investigate the structural change of HSA. It is well-known that the synchronous fluorescence gives the characteristic information of tyrosine residues or tryptophan residues when the D-value (△λ) between excitation wavelength and emission wavelength were stabilized at 15 or 60 nm. It can be known that the structure of HSA changed in the presence of ICA according to the increase of polarity of tryptophan residues(5nm shift in position of emission maximum) at△λ=60nm while the changeless of tyrosine residues at△λ=15nm. The result of three-dimensional fluorescence spectra and contour spectra of HSA in the presence of ICA also proved that the structure of HSA changed significantly.Chapter 3. Studies on the interaction between puerarin and bovine serum albumin as well as effect of the coexistent ions on the reactionPuerarin (PUE) is the substance extracted from pueraria lobatas. The interaction between puerarin and BSA in physiological condition was studied by fluorescence and UV-visible spectrophotometry. The quenching constant and the binding constant are 7.29×10⁴ L·mol^-1 and 5.04×10⁴ L·mol^-1 according to the equation of Stern-Volmer and Lineweaver-Burk, respectively. The experiment demonstrated that the quenching mechanism for PUE with BSA was the static quenching process which quenching rate constant(K_q=7.29×10¹²L·mol^-1·S^-1) was greater than the maximum scatter collision quenching constant of various quenchers with the biopolymer(2×10¹⁰L·mol^-1·s^-1). The binding distance between donor(BSA) and acceptor(PUE) was calculated to be 3.53 nm which based on the theory of Forster spectroscopy energy transfer, the energy transfer efficiency (E=0.1355) and the overlap integral of the fluorescence emission spectrum of BSA and the absorption spectrum of PUE(J=8.05×10^-15cm³Â·L·mol^-1). From the synchronous fluorescence spectra of BSA, it can be known that the structure of BSA changed in the presence of PUE according to the increase of polarity of tryptophan residues(5nm shift in position of emission maximum) at△λ=60 nm while the changeless of tyrosine residues at△λ= 15nm. In addition, the competition reaction for BSA between Zn²⁺, Cu²⁺, Mg²⁺, Ca²⁺and PUE were discussed, the results showed that the binding ability between BSA and PUE increased in the presence of Zn²⁺, Cu²⁺ and Mg²⁺, so there is longer effect of drugs in bodies; The binding ability decreased in the presence of Ca²⁺, so there is stronger effect of drugs in quite short time. Further more, the quantitative analysis of PUE was carried out by the synchronous fluorescence spectrophotometry at△λ=110nm, the LOD is 0.0228μg·ml^-1.Chapter 4. Studies on the interaction between enrofloxacin and bovine serum albumin by fluorescence spectrometryEnrofloxacin (ENR) is the third generation of fluoroquinolone derivatives. In this section, the interaction between enrofloxacin and BSA in physiological condition was studied by fluorescence and UV-visible spectrophotometry. The experiment demonstrated that the fluorescence of BSA could be quenched by ENR. The quenching mechanism for ENR with BSA was the static quenching process which quenching rate constant(K_q=3.10×10¹³ L·mol^-1·s^-1) was greater than the maximum scatter collision quenching constant of various quenchers with the biopolymer(2×10¹⁰ L·mol^-1·s^-1), the binding constant is 2.60×10⁵ L·mol^-1 and the binding site is 1.07. The binding distance between donor(BSA) and acceptor(ENR) was calculated to be 2.57nm which based on the theory of Forster spectroscopy energy transfer, the energy transfer efficiency (E=0.279) and the overlap integral of the fluorescence emission spectrum of HSA and the absorption spectrum of ENR(J=4.04×10^-15 cm³Â·L·mol^-1). In addition, the competition reaction for BSA between Cu²⁺, Zn²⁺ and ENR were discussed, it seems that the binding constant decreased between BSA and ENR while Cu²⁺ and Zn²⁺ existed, so the drug will be released more quickly in boides and the stronger effect of drugs in quite short time. Chapter 5. Studies on the identification of alpinetin and calf thymus DNAThe identification between ALP and DNA in physiological condition was investigated by fluorescence and UV-visible spectrophotometry. The experiment demonstrated that the fluorescence of ALP could be quenched by DNA, the binding mechanism was proved to be the single static quenching process according to the deescalating binding constant (3.288×10³, 2.923×10³ and 2.467×10³L·mol^-1) along with the escalating temperatures (25℃, 32℃and 39℃). From the UV-visible spectra between ALP and DNA and the fluorescence spectra of EB-DNA, the results showed that the UV-visible spectra of ALP did not changed in the presence of DNA and the fluorescence intensity of EB-DNA did not decreased in the presence of ALP, further more, the T_m of DNA(75.8℃) and the T_m of ALP-DNA(76.2℃) almost be the same with each other, so it was concluded that there was not intercalation binding mode between ALP and DNA. A kind of groove binding mode may existed according to the effect of I. For further study, the result showed that the electrostatic binding mode was existed between ALP and ss-DNA. At last, the effect of NaCl was investigated, the experiment demonstrated that the fluorescence intensity of ALP-DNA decreased by the increasing concentration of NaCl, it proved that the groove binding mode was existed again. In a word, we can get a conclusion that ALP can interact with DNA, and the groove binding mode was mainly existed.Chapter 6. Studies on the interaction of enrofloxacin and calf thymus DNA by spectroscopic methodsThe interaction of ENR and calf thymus DNA in physiological condition was investigated by fluorescence and UV-visible spectrophotometry. It demonstrated that the binding mechanism was the single static quenching process according to the UV-visible spectra of ENR and BSA and the quenching rate constant(K_q=6.47×10¹² L·mol^-1·s^-1), the binding constant is 2.73×10⁵ L·mol^-1, and the binding site is 1.12. Through the effect of neutral red (NR) which used to be as a fluorescent probe, the result showed that NR could be permuted from NR-DNA, that means there was intercalation binding mode existed between ENR and DNA. In addition, the effect of H₂PO₄^- was also investigated, the result showed that electrostatic binding mode was existed because the fluorescence intensity of ENR-DNA decreased in the presence of the H₂PO₄^-. For further study, it proved that there were electrostatic binding mode and intercalation binding mode existed together according to the effect of I^-.